Project Summary/Abstract During nervous system development and post-injury regeneration, nerve pathways are formed by migrating neuronal axons which are guided by extracellular cues. Although many of the major signaling pathways that regulate axon guidance have been identified, our understanding of the mechanisms by which individual genes influence specific axon guidance outcomes remains limited. In animals with complex nervous systems, such as insects and mammals, individual ligands and receptors from conserved signaling pathways can promote diverse or even opposing outcomes in different populations of neuronal axons. This proposal uses the Drosophila Robo2 receptor as a model to address the question of how individual genes can specify diverse axon guidance outcomes. Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors, and Robo2 controls at least three distinct axon guidance outcomes in the Drosophila embryonic central nervous system (CNS). The proposed research takes advantage of the molecular and genetic tools available in Drosophila to dissect the molecular and cellular mechanisms underlying the diverse axon guidance activities of Robo2. Specifically, the project will use a structure/function gene modification approach to characterize the functional domains within Robo2 that contribute to each of its axon guidance activities (Aim 1), test candidate mechanisms that may account for Robo2's uncharacterized role in promoting longitudinal pathway formation (Aim 2), and investigate how the transcriptional regulation of robo2 expression in specific neuronal populations contributes to its diverse roles in axon guidance (Aim 3). The proposed research will provide insight into how individual axon guidance genes can specify diverse developmental outcomes, identify potentially evolutionarily conserved mechanisms that may operate in other animals, including humans, where members of the Robo receptor family are also key regulators of axon guidance, and may suggest novel strategies for restoring proper regulation of axon guidance in the contexts of nervous system injury, repair, and regeneration.